Antibacterial Effect of Oral Care Gel-Containing Hinokitiol and 4-isopropyl-3-methylphenol Against Intraoral Pathogenic Microorganisms

Objective Deterioration of oral hygiene is closely related to increase severity and mortality of corona virus disease-19 (COVID-19), and also contribute to the development of various diseases such as aspiration pneumonia or Alzheimer’s. Oral care is attracting high interest in Japan, which has entered a super-aging society. In this study, we aimed to investigate whether commercially available Hinora® (HO) that oral care gels-containing hinokitiol and 4-isopropyl-3-methylphenol (IPMP) have biofilm formation inhibitory and antibacterial activities against various intraoral pathogen microorganisms. Method Candida spp., Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Pseudomonas aeruginosa were selected during the study period, all which were analyzed using antimicrobial disc, microorganism turbidity, and crystal violet assays. In addition, the germ tube test using C. albicans was performed with a modification of Mackenzie’s method. Images for morphological observation of the germ tubes were acquired with an inverted microscope. For comparison between products, we used Refrecare® (RC), which contains only hinokitiol (not containing IPMP). Results All the intraoral pathogenic microorganisms showed drug susceptibility against undiluted form HO and/or RC. In particular, HO was more effective at lower concentrations than RC. In the HO-added group, inhibition circles were observed in all bacteria except P. aeruginosa when added at a concentration of 0.5 g/mL or more. The optical density values at 590 nm (crystal violet) and/or 600 nm (microorganism turbidity) of all the fungi and bacteria were significantly lower when cultured in medium with HO. Inhibition of growth or biofilm formation was observed when HO was added at a concentration of 0.05 g/mL or higher. To investigate the action mechanism of HO, germ tube tests were performed in C. albicans. The results show that culturing C. albicans in soyabean-casein digest broth with HO (0.05 g/mL) significantly suppressed germ tube formation. Conclusions These data suggest that oral care gel-containing hinokitiol and IPMP has strong biofilm formation inhibitory, antifungal and antibacterial effects against Candida fungi and multiple intraoral pathogenic microorganisms. Therefore, it may be a promising treatment option for oral infections.


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Oral infections such as oral candidiasis and periodontal disease is a series of 52 polymicrobial infection that affect the oral mucosa and tooth root [1]. Deterioration of oral 53 hygiene leads to intraorally colonization of fungi or bacteria, it has been reported to 54 contribute to systemic diseases such as gingivitis, aspiration pneumonia, deep mycosis, or 55 increased severity and mortality in the historical pandemic of corona virus disease-19 56 (COVID-19) [2][3][4]. It is suggested that aspiration pneumonia, which is often seen in the 4 57 elderly, is caused by oral bacteria growing in the oral cavity. Oral care is attracting high 58 interest in Japan, which has entered a super-aging society. Opportunistic infectious 59 pathogenic microorganisms such as Candida albicans are known to easily form multi-60 species biofilms coaggregation on the surfaces of teeth and dentures [3,5]. Candida spp.    Evaluation (NBRC, Chiba, Japan). All bacteria and fungi, except A. 104 actinomycetemcomitans, were cultured under aerobic conditions, and A. 105 actinomycetemcomitans was cultured under anaerobic conditions (5 % CO 2 , 0 % O 2 ), using 106 Anaero Pouch Kenki (Mitsubishi Gas Chemicals Co., Ltd., Tokyo, Japan).

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Antimicrobial disc diffusion assay 119 Each fungus and bacterium were inoculated into saline solutions to a concentration of 120 1 × 10 6 or 1 × 10 8 CFU/mL and spread evenly on SCD agar medium. A sterilized paper 121 disc with a diameter of 8 mm (ADVANTEC Toyo, Co., Ltd., Tokyo, Japan) coated with 122 oral care gel-containing medium was placed on the SCD agar medium, and the fungus or 123 bacterium was cultured at 37°C for 24 h. Paper discs were coated with 80 μL of SCD broth and SCD broth containing HO (0.1, 125 0.5, and 5 g/mL and undiluted form) or RC (0.1, 0.5, and 5 g/mL and undiluted form). A 126 paper disc coated with oral care gel-containing medium was used after 2-3 h of air drying 127 in a clean work station.

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Microorganism turbidity and crystal violet assays 129 As described above, each fungus and bacterium were cultured at 37 °C for 24-48 h in 130 SCD broth and SCD broth containing HO (0.05 g/mL, 0.125 g/mL) or RC (0.05 g/mL,  with an Olympus CKX41 camera (Olympus Co., Ltd., Tokyo, Japan).

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Statistical analysis 149 The statistical significance of the differences in the ratio of biofilm formation between 150 non-treated bacterial pathogens and HO-and RC-treated bacterial pathogens was examined 151 using Dunnett's test. The statistical significance level was considered to be statistically 152 significant when P < 0.05.

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The inhibitory zone formed by the undiluted solution of HO and RC for each Candida 155 fungus was larger than that of the control group (Fig. 1). The inhibition zone was larger for 156 HO than for RC, with diameters of 24 mm for C. albicans (Fig. 1A), 25 mm for C. glabrata 157 (Fig. 1B), 25 mm for C. krusei (Fig. 1C), 24 mm for C. parapsilosis (Fig. 1D), and 23 mm 158 for C. tropicalis (Fig. 1E). In addition, all oral infection-causing bacteria used in the 159 experiments formed inhibitory zones, and the maximum zone of inhibition was 31 mm of S. 160 aureus in the HO-added group. Table 1 shows the details of the inhibition zone diameter. In 161 the HO-added group, inhibition circles were observed in all bacteria except P. aeruginosa 162 at a concentration of 0.5 g/mL or higher. In the RC-added group, inhibition circles were 163 observed in all bacteria except C. krusei at a concentration of 5 g/mL or higher (Table 1). 164 We next cultured various bacteria in SCD broth containing HO or RC (final 0.05, 0.25 165 g/mL) for 24 h, and bacterial growth was measured using spectrophotometry by absorbance 166 at OD 600 nm. The value of OD 600 nm was significantly lower when cultured in SCD 9 167 broth containing HO or RC compared to the control (Fig. 2). These growth suppression 168 effects were observed when the concentration of HO or RC was 0.05 g/mL or higher. 169 Subsequently, we investigated whether these oral care gels had biofilm formation 170 inhibitory activity against various bacteria using crystal violet assays. The addition of HO 171 inhibited biofilm formation of all the oral infection-causing bacteria used (Fig. 3). The 172 inhibitory effect of HO was observed at 0.05 g/mL or higher. On the other hand, RC 173 showed a biofilm formation inhibitory effect against bacteria other than A. 174 actinomycetemcomitans. The inhibitory effect of RC was observed at 0.05 g/mL or higher.

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Finally, we conducted a germ tube test of C. albicans on a HO-containing SCD broth 176 to investigate the mechanism of action of HO, which had an excellent ability to inhibit 177 biofilm formation. The results show that the addition of HO significantly suppressed the 178 formation of germ tubes in C. albicans (Fig. 4). Germ tube formation was inhibited even at 179 a concentration as low as 0.05 g/mL of HO.  (Table 1). In the HO-added group, inhibition circles were observed in 192 all bacteria except P. aeruginosa when added at a concentration of 0.5 g/mL or more. In the 193 RC-added group, inhibition circles were observed in all bacteria except C. krusei when 194 added at a concentration of 5 g/mL or more (Table 1). 195 In addition, various fungi and bacteria described above were cultured in SCD broth those of cultures without HO or RC (Fig. 2). Growth inhibition was observed when HO or 201 RC was added at a concentration of 0.05 g/mL or higher (Fig. 2). These results indicate that 202 HO and RC showed growth inhibitory effects on the microorganisms used in this study.

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Furthermore, the results of the disc diffusion assay and culture in SCD liquid medium 204 showed that HO tended to have higher antibacterial activity than that of RC. Hinokitiol   HO is presumed to suppress biofilm formation by inhibiting the adhesion of C. 246 albicans to the tooth surface. In the future, it will be necessary to investigate in detail the 247 mechanism of HO and its mechanism of action in other microorganisms as well. In 248 addition, given that these oral care gels exerted a biofilm formation inhibitory effect and an 249 antibacterial effect against Candida spp. at a low concentration (0.05 g/mL), the effects of 250 the levels used for oral care should be investigated. Moreover, the optimal use of these gels, 251 that is, whether to use these when brushing teeth, to gargle these with water after brushing 252 teeth, and so on, should be considered in future studies.

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In conclusion, oral care gel-containing hinokitiol were found to have biofilm